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Water cycling across the membrane transporters is considered a hallmark of cellular metabolism and it could be of high diagnostic relevance in the characterization of tumors and other diseases. The method relies on the response of intracellular proton exchanging molecules to the presence of extracellular Gd‐based contrast agents (GBCAs). Paramagnetic GBCAs enhances the relaxation rate of water molecules in the extracellular compartment and, through membrane exchange, the relaxation enhancement is transferred to intracellular molecules. The effect is detected at the MRI‐CEST (Magnetic Resonance Imaging ‐ Chemical Exchange Saturation Transfer) signal of intracellular proton exchanging molecules. The magnitude of the change in the CEST response reports on water cycling across the membrane. The method has been tested on Red Blood Cells and on orthotopic murine models of breast cancer with different degree of malignancy (4T1, TS/A and 168FARN). The distribution of voxels reporting on membrane permeability fits well with the cells' aggressiveness and acts as an early reporter to monitor therapeutic treatments. [ABSTRACT FROM AUTHOR]

Water cycling across the membrane transporters is considered a hallmark of cellular metabolism and it could be of high diagnostic relevance in the characterization of tumors and other diseases. The method relies on the response of intracellular proton exchanging molecules to the presence of extracellular Gd‐based contrast agents (GBCAs). Paramagnetic GBCAs enhances the relaxation rate of water molecules in the extracellular compartment and, through membrane exchange, the relaxation enhancement is transferred to intracellular molecules. The effect is detected at the MRI‐CEST (Magnetic Resonance Imaging ‐ Chemical Exchange Saturation Transfer) signal of intracellular proton exchanging molecules. The magnitude of the change in the CEST response reports on water cycling across the membrane. The method has been tested on Red Blood Cells and on orthotopic murine models of breast cancer with different degree of malignancy (4T1, TS/A and 168FARN). The distribution of voxels reporting on membrane permeability fits well with the cells' aggressiveness and acts as an early reporter to monitor therapeutic treatments. [ABSTRACT FROM AUTHOR]

The aquaporin‐4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults, such as traumatic injury or stroke. Lack of specific and therapeutic AQP4 inhibitors highlights the need to explore alternative routes to control the water permeability of glial cell membranes. The cell surface abundance of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen levels and tonicity, suggesting a role for vesicular trafficking in its translocation to and from the cell surface. However, the molecular mechanisms of AQP4 trafficking are not fully elucidated. In this work, early and recycling endosomes were investigated as likely candidates of rapid AQP4 translocation together with changes in cytoskeletal dynamics. In transiently transfected HEK293 cells a significant amount of AQP‐eGFP colocalised with mCherry‐Rab5‐positive early endosomes and mCherry‐Rab11‐positive recycling endosomes. When exposed to hypotonic conditions, AQP4‐eGFP rapidly translocated from intracellular vesicles to the cell surface. Co‐expression of dominant negative forms of the mCherry‐Rab5 and ‐Rab11 with AQP4‐eGFP prevented hypotonicity‐induced AQP4‐eGFP trafficking and led to concentration at the cell surface or intracellular vesicles respectively. Use of endocytosis inhibiting drugs indicated that AQP4 internalisation was dynamin‐dependent. Cytoskeleton dynamics‐modifying drugs also affected AQP4 translocation to and from the cell surface. AQP4 trafficking mechanisms were validated in primary human astrocytes, which express high levels of endogenous AQP4. The results highlight the role of early and recycling endosomes and cytoskeletal dynamics in AQP4 translocation in response to hypotonic and hypoxic stress and suggest continuous cycling of AQP4 between intracellular vesicles and the cell surface under physiological conditions. [ABSTRACT FROM AUTHOR]

The canonical autophagy pathway in mammalian cells sequesters diverse cytoplasmic cargo within the double membrane autophagosomes that eventually convert into degradative compartments via fusion with endolysosomal intermediates. Here, we report that autophagosomal membranes show permeability in cells lacking principal ATG8 proteins (mATG8s) and are unable to mature into autolysosomes. Using a combination of methods including a novel in vitro assay to measure membrane sealing, we uncovered a previously unappreciated function of mATG8s to maintain autophagosomal membranes in a sealed state. The mATG8 proteins GABARAP and LC3A bind to key ESCRT‐I components contributing, along with other ESCRTs, to the integrity and imperviousness of autophagic membranes. Autophagic organelles in cells lacking mATG8s are permeant, are arrested as amphisomes, and do not progress to functional autolysosomes. Thus, autophagosomal organelles need to be maintained in a sealed state in order to become lytic autolysosomes. Synopsis: Mammalian ATG8s (mATG8s) interact with ESCRT‐I components to maintain autophagosomal membranes in a sealed, nonporous state that enables autophagic intermediates to progress into lytic organelles. mATG8s bind and recruit ESCRT components to maintain autophagic membranes in a sealed state impermeable to solutesBoth LC3 and GABARP subsets of mATG8s are requiredThe most widely used marker of autophagy, LC3B, is not involved in these processesAutophagic organelles not maintained in a sealed state are arrested at the amphisomal stage [ABSTRACT FROM AUTHOR]

Objectives: Tight junctions (TJs) are involved in the regulation of salivary secretion via paracellular pathway. Botulinum toxin type A (BTXA) is widely used for the treatment of hypersecretion diseases such as sialorrhea. This study aimed to investigate the role of TJs in BTXA‐inhibited secretion of the submandibular gland (SMG). Materials and Methods: BTXA was injected into the SMGs of rats, and the same amount of saline was injected as a control. Western blot, real‐time PCR, and immunofluorescence staining were used to detect the expression and distribution of TJ proteins. Paracellular permeability was evaluated using the transepithelial electrical resistance (TER) measurements and fluorescent tracer detection in BTXA‐stimulated SMG‐C6 cells. Results: BTXA injection into the SMGs of rats led to increased expression of claudin (Cldn) −1 and Cldn3. Immunofluorescence staining showed no significant changes in the distribution of TJ proteins. In vitro, BTXA increased the TER values and significantly reduced the permeability of fluorescent tracer, suggesting that BTXA decreased the paracellular permeability. The expression levels of Cldn1, Cldn3, and Cldn4 were upregulated after BTXA treatment. Conclusion: The expression of TJ proteins changed in both animal models and SMG‐C6 cells after BTXA treatment, which may contribute to the inhibition of salivary secretion. [ABSTRACT FROM AUTHOR]

Methods to follow in real time complex processes occurring along living cell membranes such as cell permeabilization are rare. Here, the terahertz spectroscopy reveals early events in plasma membrane alteration generated during photodynamic therapy (PDT) protocol, events which are not observable in any other conventional biological techniques performed in parallel as comparison. Photodynamic process is examined in Madin‐Darby canine kidney cells using Pheophorbide (Pheo) photosensitizer alone or alternatively encapsulated in poly(ethylene oxide)‐block‐poly(ε‐caprolactone) micelles for drug delivery purpose. Terahertz spectroscopy (THz) reveals that plasma membrane permeabilization starts simultaneously with illumination and is stronger when photosensitizer is encapsulated. In parallel, the exchange of biological species is assessed. Over several hours, this conventional approach demonstrates significant differences between free and encapsulated Pheo, the latter leading to high penetration of propidium iodide, Na+ and Ca2+ ions, and a high level of leakage of K+, ATP, and lactate dehydrogenase. THz spectroscopy provides, in a single measurement, the relative number of defects per membrane surface created after PDT, which is not achieved by any other method, providing early, sensitive real‐time information. THz spectroscopy is therefore a promising technique and can be applied to any biological topic requiring the examination of short‐term plasma membrane permeabilization. [ABSTRACT FROM AUTHOR]

Different types of natural K+ channels share similar core modules and cation permeability characteristics. In this study, we have developed novel artificial K+ channels by rebuilding the core modules of natural K+ channels in artificial systems. All the channels displayed high selectivity for K+ over Na+ and exhibited a selectivity sequence of K+≈Rb+ during the transport process, which is highly consistent with the cation permeability characteristics of natural K+ channels. More importantly, these artificial channels could be efficiently inserted into cell membranes and mediate the transmembrane transport of K+, disrupting the cellular K+ homeostasis and eventually triggering the apoptosis of cells. These findings demonstrate that, by rebuilding the core modules of natural K+ channels in artificial systems, the structures, transport behaviors, and physiological functions of natural K+ channels can be mimicked in synthetic channels. [ABSTRACT FROM AUTHOR]

Different types of natural K+ channels share similar core modules and cation permeability characteristics. In this study, we have developed novel artificial K+ channels by rebuilding the core modules of natural K+ channels in artificial systems. All the channels displayed high selectivity for K+ over Na+ and exhibited a selectivity sequence of K+≈Rb+ during the transport process, which is highly consistent with the cation permeability characteristics of natural K+ channels. More importantly, these artificial channels could be efficiently inserted into cell membranes and mediate the transmembrane transport of K+, disrupting the cellular K+ homeostasis and eventually triggering the apoptosis of cells. These findings demonstrate that, by rebuilding the core modules of natural K+ channels in artificial systems, the structures, transport behaviors, and physiological functions of natural K+ channels can be mimicked in synthetic channels. [ABSTRACT FROM AUTHOR]